
16.3 小分子递质被主动吸收到囊泡中
372 Part III / Synaptic Transmission
200 微米
4 GAD65–GFP 5 γ-氨基丁酸
I
I
II–III
II–III
IV
IV
1 GAD65–GFP 2 GAD65
3 叠加
6 叠加
皮层
2 GAD
1 GAT-1
Figure 16–3 Techniques for visualizing chemical
messengers.
A.A light-microscope section of the hippocampus of a rat.
1. In situ hybridization using a probe for the mRNA encod-
ing GAT-1, a GABA transporter. The probe was end-labeled
with α-
35
S-dATP and visualized by clusters of silver grains
in the overlying autoradiographic photographic emulsion.
2. In situ hybridization of the mRNA for glutamic acid
decarboxylase (GAD), the specific biosynthetic enzyme
for GABA, was carried out with an oligonucleotide probe
linked to the enzyme alkaline phosphatase. The GAD
probe was visualized by accumulation of colored alkaline
phosphatase reaction product in the cytoplasm. Neurons
expressing both GAT-1 and GAD transcripts were labeled
by silver grains and the phosphatase reaction, respec-
tively, and are indicated by circles enclosing cells bodies
that contain both labels. (Used with permission of Sarah
Augood.)
B.Images of neocortex from a GAD65-GFP transgenic
mouse in which green fluorescent protein (GFP) is
expressed under the control of the GAD65 promotor. GFP
is co-localized with GAD65 (1–3) and GABA (4–6) (both
detected by indirect immunofluorescence) in neurons in
the different layers. Most of the GFP-positive neurons
are immunopositive for GAD65 and GABA (arrows show
selected examples). Scale bar = 100 μm.(Adapted, with
permission, from López-Bendito et al. 2004. Copyright ©
2004 Oxford University Press.)
Powerful histochemical techniques are available for
detecting both small-molecule transmitter substances
and neuroactive peptides in histological sections of
nervous tissue.
Catecholamines and serotonin, when reacted with
formaldehyde vapor, form fluorescent derivatives. In an
early example of transmitter histochemistry, the Swedish
neuroanatomists Bengt Falck and Nils Hillarp found
that the reaction can be used to locate transmitters with
fluorescence microscopy under properly controlled
conditions.
Because individual vesicles are too small to be
resolved by the light microscope, the exact position of
the vesicles containing the transmitter was inferred by
comparing the fluorescence under the light microscope
with the position of vesicles under the electron micro-
scope. A number of fluorescent false transmitters, par-
ticularly those that mimic catecholamines, are substrates
for plasma membrane and/or vesicular transporters,
enabling their use to label vesicles and assess their turn-
over in living tissue. In addition, a variety of genetically
expressed neurotransmitter reporters based on green
fluorescent protein can be used to detect extracellular
levels of neurotransmitters.
Histochemical analysis can be extended to the ultra-
structure of neurons under special conditions. Fixation
of nervous tissue in the presence of potassium perman-
ganate, chromate, or silver salts, or the dopamine analog
Box 16–2 Detection of Chemical Messengers and Their Processing Enzymes Within Neurons
Kandel-Ch16_0358-0378.indd 372 18/01/21 5:46 PM
图 16.3.2: 可视化化学信使的技术。A. 大鼠海马体的光学显微镜切片。1. 使用编码γ-氨基丁酸转运蛋白 GAT-1 的
信使核糖核酸的探针的原位杂交。用 α-35S dATP 对探针进行末端标记,并通过覆盖的放射自显影照相乳剂中的
银颗粒簇进行观察。2. 用与碱性磷酸酶连接的寡核苷酸探针对谷氨酸脱羧酶的信使核糖核酸进行原位杂交,谷
氨酸脱羧酶是 γ-氨基丁酸的特异性生物合成酶。谷氨酸脱羧酶探针通过细胞质中有色碱性磷酸酶反应产物的积
累而显现。表达 GAT-1 和谷氨酸脱羧酶转录物的神经元分别用银粒和磷酸酶反应标记,并用包含这 2 种标记的
细胞体周围的圆圈表示。B. 来自谷氨酸脱羧酶 65-绿色荧光蛋白转基因小鼠的新皮层的图像,其中绿色荧光蛋白
在谷氨酸脱羧酶 65 启动子的控制下表达。绿色荧光蛋白与谷氨酸脱羧酶 65(1-3)和γ-氨基丁酸(4-6)(均通过
间接免疫荧光检测)共同定位于不同层的神经元中。大多数绿色荧光蛋白阳性神经元对谷氨酸脱羧酶 65 和γ-氨
基丁酸呈免疫阳性(箭头显示所选实例)。比例尺 =100 微米。
Chapter 16 / Neurotransmitters 373
Figure 16–4 Electron-opaque gold particles linked to
antibody are used to locate antigens in tissue at the
ultrastructural level.The electron micrograph shows a
section through the cell body of an Aplysia bag cell. Bag
cells control reproductive behavior by releasing a group of
neuropeptides cleaved from the egg-laying hormone (ELH)
precursor. The cells contain several kinds of dense-core
vesicles. The cell shown here was treated with two anti-
bodies against different amino acid sequences contained
in different regions of the ELH precursor. One antibody
was raised in rabbits and the other in rats. These antibod-
ies were detected with anti-rabbit or anti-rat immunoglobu-
lins (secondary antibodies) raised in goats. Each secondary
antibody was coupled to colloidal gold particles of a dis-
tinct size. Vesicles identified by antigen 1 (labeled with the
smaller gold particles) are smaller than vesicles identified
by antigen 2 (labeled with the larger gold particles), indicat-
ing that the specific fragments cleaved from the precursor
are localized in different vesicles.(Reproduced, with per-
mission, from Fisher et al. 1988.)
抗原
1
抗原
2
240 纳米
囊泡含有:
5-hydroxdopamine, which forms an electron-dense
product, intensifies the electron density of vesicles
containing biogenic amines and thus reveals the large
number of dense-core vesicles that are characteristic of
aminergic neurons.
It is also possible to identify neurons that express
the gene for a particular transmitter enzyme or peptide
precursor. Many methods for detecting specific mRNAs
depend on nucleic acid hybridization. One such method
is in situ hybridization.
Two single strands of a nucleic acid polymer will pair
if their sequence of bases is complementary. With in situ
hybridization, the strand of noncoding DNA (the negative
or antisense strand or its corresponding RNA) is applied
to tissue sections under conditions suitable for hybridizing
with endogenous (sense) mRNA. If the probes are radiola-
beled, autoradiography reveals the locations of neurons
that contain the complex formed by the labeled comple-
mentary nucleic acid strand and the mRNA.
Hybrid oligonucleotides synthesized with nucle-
otides containing base analogs tagged chemically,
fluorescently, or with antibodies can be detected his-
tochemically. Multiple labels can be used at the same
time (Figure 16–3A). RNAscope, a more recent mRNA
hybridization method, allows for simultaneous detec-
tion of different mRNAs with lower background and
single-molecule sensitivity. Another approach to detect-
ing the synthetic proteins involves viral or transgenic
expression of proteins fused to variants of green fluores-
cent protein (Figure 16–3B).
Transmitter substances can also be detected using
immunohistochemical techniques. Amino acid trans-
mitters, biogenic amines, and neuropeptides have a pri-
mary amino group that becomes covalently fixed within
the neurons; this group becomes cross-linked to proteins
by aldehydes, the usual fixatives used in microscopy for
immunohistochemical techniques.
Specific antibodies against the transmitter sub-
stances are necessary. Antibodies specific to seroto-
nin, histamine, and many neuroactive peptides can be
detected by a second antibody (in a technique called
indirect immunofluorescence). As an example, if the first
antibody is rabbit-derived, the second antibody can be
goat antibody raised against rabbit immunoglobulin.
These commercially available secondary antibod-
ies are tagged with fluorescent dyes and used under the
fluorescence microscope to locate antigens in regions of
individual neurons—cell bodies, axons, and presynaptic
release sites (Figure 16–3).
Immunohistochemical techniques are also used
with electron microscopy to locate chemical transmit-
ters in the ultrastructure of neurons. Such techniques
(continued)
Kandel-Ch16_0358-0378.indd 373 18/01/21 5:46 PM
图 16.3.3: 与抗体相连的电子不透明金颗粒用于在超微结构水平上定位组织中的抗原。电子显微照片显示了穿过
海兔袋细胞的细胞体的截面。囊细胞通过释放一组从产卵激素前体切割而来的神经肽来控制生殖行为。细胞含
有几种致密的核心囊泡。用针对产卵激素前体的不同区域中所含的不同氨基酸序列的 2 种抗体处理此处显示的
细胞。一种抗体在兔子身上产生,另一种在大鼠身上产生。用山羊饲养的抗兔或抗大鼠免疫球蛋白(第二抗体)
检测这些抗体。每种二级抗体都与不同大小的胶体金颗粒偶联。由抗原 1 鉴定的囊泡(用较小的金颗粒标记)小
于由抗原 2 鉴定的囊袋(用较大的金颗粒标记),这表明从前体切割的特异性片段定位在不同的囊泡中。
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